Analysis of biomolecules is a challenge due to the complex nature of biological samples. Most biological samples contain a diverse range of analytes with different biochemical functionalities, molecular sizes and masses as well as presence in varying abundances. Furthermore, these can also be present in various physical manifestations such as liquids, vapors/gaseous phases containing volatiles, solids and various forms of soft matter such as tissues, emulsions, networks, composites etc. Thus, maneuvering a given biological sample to analyze and understand the biochemical components present therein is an enormous task. Such information is of vital importance in healthcare/medical applications and numerous other commercial applications that are non medical/healthcare in nature.
One of the approaches followed in such situations is to study the sample profile and simplify it, but this may result in compromising the scope of the analysis. Also currently, separation, isolation and detection are performed separately moving from one platform to another severely compromising analytical performance.
‘Matrix assisted laser desorption/ionization (MALDI) mass spectrometry’ is a useful and popular analytical tool for large molecules such as peptides, polymers and proteins. However, due to inherent peaks from the ‘matrix’ that interfere in the low molecular region, MALDI is quite unpopular for small molecule analysis. In MALDI MS, all the analytes including the matrix itself are detected. Suspected ion suppression from the matrices has also been considered to be a major bottleneck for small molecule analysis using MALDI MS.
Laser desorption ionization mass spectrometry (LDI MS) is commonly referred to the techniques, which do not use organic matrices (unlike MALDI MS). The consequence of this is that smaller molecules which cannot be analyzed mass spectrometrically using MALDI MS can now be analyzed along with larger molecules such as peptides. It must be emphasized that all the prior art using LDI or MALDI describe analysis of both small and large molecular weight analytes and none of these refer or imply “preferential” or “selective” analysis over the other classes of analytes. Thus prior art on LDI MS even if illustrating small molecule analysis do not make any reference to “selectivity” or “preference” over the rest of the molecules and quite often demonstrate the larger utility of LDI for both smaller and larger molecular weight analytes. A few specific examples of the prior art are detailed below in this regard.
To overcome the limitations and disadvantages of MALDI MS, a wide range of materials have been demonstrated for use as alternatives to the organic matrices in LDI MS. These include polymers, surfactants, activated carbon, carbon nanotubes and inorganic materials (like germanium nanodots (Seino, 2007 #33), platinum nanoflowers (Kawasaki, Yonezawa et al. 2007), metal oxide nanoparticles (Kinumi, Saisu et al. 2000), silicon nanowires (Go, Apon et al. 2005), metal oxide porous films (Chen and Chen 2004) etc.). The use of metal oxide particles and films being the most preferred as one could perform a ‘matrix-free’ analysis in real sense by removing/reducing the contribution from the matrix in the small molecule region (m/z<700).
One example of ‘matrix-free’ analysis is porous silicon (not silica), popularly known as DIOS (desorption ionization on porous silicon), which was investigated for its use in mass spectrometry as an alternative to MALDI MS. Wafers of silicon were produced and commercialized as ‘DIOS target plates’ for the analysis of proteins, peptides and many other analytes using laser desorption/ionization mass spectrometry (Siuzdak, Buriak et al. 2000) (Wei, Buriak et al. 1999) (Shen, Thomas et al. 2001) (Thomas, Shen et al. 2001). It is believed that the silicon wafer “softly” transmits the UV laser energy to the analytes leading to their desorption and ionization followed by mass spectral identification, usually in a ‘time-of-flight’ tube where analytes traveling in a tube with applied electrical potential get separated by virtue of their mass by charge ratios (m/z). This material and the method is universal and does not discriminate or offer selectivity between various types of analytes. The primary objective of this prior art is to provide an alternative to MALDI MS. Thus in this example described DIOS enables the detection of small as well as large MW analyte detection minus the ‘matrix’ interference.
U.S. Pat. No. 6,958,480 claims a method of performing ‘matrix-free’ laser desorption/ionization mass spectrometry using a nanocomposite comprising mesoporous silica thin film on porous silicon, glass etc. This composite film is prepared by dispersing a silica precursor (tetraethyl orthosilicate TEOS) in a surfactant solution that is spread on yet another material, a silica wafer or a glass slide in this embodiment, to make it into a composite material in the form of a thin film. The final nanocomposite thin film is obtained by removing the surfactant employed as a template by means of exposing the films to deep UV light or by calcination at elevated temperatures. An analyte is then placed on this composite thin film, which is subsequently subjected to laser desorption/ionization mass spectrometry as described above. The thickness of the film is in the range of 70-300 nm with pore sizes of 1-50 nm. This nanocomposite film presents better performance characteristics over the above mentioned DIOS plates, but is used essentially for the same purpose of universally analyzing samples. Its advantage is as a superior method relative to the traditional MALDI MS. Thus, this nanocomposite based mesoporous technology also enables the detection of small as well as large MW analytes in a given sample minus any interference from the matrix. The invention does not disclose any selective analysis or ionization resulting from the material or the method. The inventors in the above mentioned patent also disclosed that peptides such as Angiotensin II, Bradykinin 1-7, P14R and ACTH 18-39 fragments were detected using this method wherein LDI MS was performed by placing a sample on the nanocomposite (Dattelbaum, Hicks et al. 2008). An article titled “Mesoporous silica for Desorption-Ionization Mass Spectrometry” by the same authors A. Dattelbaum et al published in Nanotech, Vol. 1, Ch. 5, pages 225-228, 2005. (Srinivas Iyer and Andrew M. Dattelbaum 2005) discloses use of mesoporous silica thin film nanocomposites for mass spectrometric analysis of both tryptophan (lower m/z) and angiotensin (peptide).
It is also noteworthy that “porous silicon” wafer alone is already covered as a patent by another group for its utility in LDI MS (no selectivity has ever been covered) that is termed as DIOS as detailed in line numbers 51 to 60 (Siuzdak et. al.). Despite this, the combination of porous silicon and mesoporous silica has been granted a patent for the same utility, which is LDI MS (U.S. Pat. No. 6,958,480).
In summary, large MW analytes can only be analyzed in the LDI MS mode using either the “organic matrix” such as 2,5-dihydroxybenzoic acid (DHB) and α-cyano hydroxy cinnamic acid α-CHCA) or the “porous silicon wafer” or “a nanocomposite thin film comprising mesoporous silica” or a few other materials.
An article titled “Selective binding and enrichment for low-molecular weight biomarker molecules in human plasma after exposure to nanoporous silica particles” by Rosa Terracciano, e.t al. published in Proteomics, Volume 6 Issue 11, pg 3243-3250, 2006, having DOI 10.1002/pmic.200500614. US 2008/0277578 A1 discloses biomarker capturing strategy based on nanoporous silica particles. The strategy as described herein comprises of exposing a plasma sample to a silica particle thereby enriching low molecular weight biomolecules (m/z 800-10,000) that are peptides and proteins followed by a separate MALDI-MS analysis of the biomolecules extracted from the silica using conventional matrices like α-cyano hydroxy cinnamic acid (α-CHCA). In this document the definition of low molecular weight molecules includes analytes molecular weight range 800 to 10,000. In this document silica porous particles (not to be mistaken with mesoporous SBA-15 prepared using template assisted synthesis) or beads are used for ONLY enrichment. Analytes are then removed for conventional MALDI MS using various matrices such as α-CHCA. It is noteworthy that various methods exist for enrichment of various classes of molecules that operate without a concurrent mass spectral dimension. A case in point is the TiO2 enrichment of phosphopeptides that is a commercial product with extensive prior art. Also, the porous silica used has non uniform pore size and not as well defined as SBA-15, The document enriches peptides and proteins with m/z 800-10,000.
The present invention/method involves selective exclusion of these peptides and proteins and detecting only the smaller analytes (MW typically less than 1000 Da). This is achieved in the present invention by a material SBA-15, which enables desorption and mass spectral analysis of small molecules from mixtures containing large analytes. Signals for the small molecule analytes are obtained directly from the material itself without the addition of an external matrix.
In summary, there is no prior art on a material, process or method using mesoporous SBA-15 that can be used for the selective mass spectrometric determination of small molecular weight (typically less than 1000 m/z) analytes while excluding large molecular weight analytes in complex mixtures.
Therefore, a quick-yet selective material and method involving minimal or no sample preparation, labeled or label-free detection that enables simultaneous mass spectral analysis is an extremely valuable tool. Such tool is significantly benefit research in biological, chemical and related sciences. Such a material and method finds wide applicability in diverse areas such as drug discovery research, proteomics, metabolomics, and in general any other application where an efficient analytical solution is needed.
With the miniaturization of mass spectrometers (Shimma S., et. al. Analytical Chemistry 2010, 82, 8456; Ouyang, Z. et. al., Analytical Chemistry 2009, 81, 2421; Kissinger, P. T. et. al., http://www.ivdtechnologv.com/article/developing-point-care-mass-spectrometer), and potential use of these instruments at point of care and point of action (such as hospitals, airports, defense applications), it is also imperative that a material enabling a method and process to selectively detect, distinguish and identify analytes of interest would be of enormous utility and commercial as well as societal importance.
The invention described herein involves (a) a process/product using pure porous material that is distinct from the prior art using various other materials or various physical forms and (b) leading to a unique utility not covered by the prior art (selective or preferential mass spectral detection of low MW molecules that are not peptides or proteins; not just LDI MS or enrichment of peptides as in the prior art).